Abstract
Poly(A)-specific ribonuclease (PARN) is a highly poly(A)-specific 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. PARN belongs to the DEDD family of nucleases, and four conserved residues are essential for PARN activity, i.e. Asp-28, Glu-30, Asp-292, and Asp-382. Here we have investigated how catalytically important divalent metal ions are coordinated in the active site of PARN. Each of the conserved amino acid residues was substituted with cysteines, and it was found that all four mutants were inactive in the presence of Mg2+. However, in the presence of Mn2+, Zn2+, Co2+, or Cd2+, PARN activity was rescued from the PARN(D28C), PARN(D292C), and PARN(D382C) variants, suggesting that these three amino acids interact with catalytically essential metal ions. It was found that the shortest sufficient substrate for PARN activity was adenosine trinucleotide (A3) in the presence of Mg2+ or Cd2+. Interestingly, adenosine dinucleotide (A) was efficiently hydrolyzed in the presence of Mn2+, Zn2+, or Co2+, suggesting that the substrate length requirement for PARN can be modulated by the identity of the divalent metal ion. Finally, introduction of phosphorothioate modifications into the A substrate demonstrated that the scissile bond non-bridging phosphate oxygen in the pro-R position plays an important role during cleavage, most likely by coordinating a catalytically important divalent metal ion. Based on our data we discuss binding and coordination of divalent metal ions in the active site of PARN.
Highlights
Poly(A)-specific ribonuclease (PARN)1 is a highly poly(A)specific 3Ј-exonuclease that efficiently degrades mRNA poly(A) tails [1,2,3,4,5,6]
In the presence of Mn2؉, Zn2؉, Co2؉, or Cd2؉, PARN activity was rescued from the PARN(D28C), PARN(D292C), and PARN(D382C) variants, suggesting that these three amino acids interact with catalytically essential metal ions
PARN Mutants with Cysteine Substitution in the Active Site Are Inactive in the Presence of Mg2ϩ but Can Be Rescued in the Presence of Soft Divalent Metal Ion—It has previously been shown that four conserved acidic amino acid residues Asp-28, Glu-30, Asp-292, and Asp-382 of PARN are essential for the catalytic activity of PARN [13]
Summary
Site-directed Mutagenesis—The cysteine mutant plasmids pE33PARN(D28C), pE33PARN(E30C), pE33PARN(D292C), and pE33PARN(D382C) were obtained from pE33PARN plasmid [13] using a QuikChange site-directed Mutagenesis kit (Stratagene) following the manufacturer’s protocol. Deadenylation reactions were carried out in buffer (25 mM HEPES, pH 7.0, 100 mM NaCl, 0.1 g/l methylated bovine serum albumin, and 2 mM of indicated divalent metal ions) at 30 °C. The reactions were stopped and the resulting products were separated by electrophoresis in 25% polyacrylamide (19:1 acrylamide/bisacrylamide)/7 M urea gels. To kinetically compare the reactions generating A2, using 5Ј-32P-endlabeled A3 or A3s-Rp A3 as substrates, deadenylation reactions were performed under single turnover conditions (0.2 nM RNA substrate, 55 nM PARN, 25 mM HEPES, pH 7.0, 100 mM NaCl, 0.1 g/l methylated bovine serum albumin, and 1 mM of indicated divalent metal ion). The products were separated by gel electrophoresis, and the total amount of A2 and A1 products was quantified at each time point using a 400S PhosphorImager (Amersham Biosciences). Calculated kobs values were based on at least three independent experiments
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